Heterotopic ossification (HO), defined as the inappropriate formation of bone in skeletal muscles and associated soft tissues, causes pain, swelling, decreased rotation of affected joints, and other health consequences. HO is a common consequence of certain surgeries and other physical traumas, and also is the defining feature of the severely debilitating condition, fibrodysplasia ossificans progressiva (FOP), a genetic disorder of dysregulated bone morphogenetic protein (BMP) signaling. Despite its prevalence and health consequences, the pathophysiology of HO is poorly understood and the progenitor cells responsible for HO have not been identified. The overarching objective of the proposed research is to identify and characterize the cellular precursors that are responsible for HO. The following hypothesis will be tested: cells of the peripheral vasculature function as osteogenic progenitor cells in response to excessive or dysregulated BMP signaling. In Aims 1 and 2, Cre/lox lineage-tracing methods will be used to determine the contribution of endothelial cells, hematopoietic cells, vascular smooth muscle, pericytes and muscle SP stem cells to HO. HO will be induced by intramuscular injection of BMP2, and the contribution of labeled cell populations to heterotopic cartilage and bone determined by immunohistochemistry using cell-specific markers. In a complementary, independent approach, a cell transplantation bioassay will assess the osteogenic potential of cellular subfractions sorted on the basis of lineage-restricted reporter gene expression, cell surface marker expression or dye exclusion properties (muscle SP). In Aim 3, a genetic mouse model for HO will be developed and characterized. The model is based on Cre recombinase-based conditional expression of the mutant Type I BMP receptor, ACVR1(R206H), which was recently found to cause FOP. In Aim 4, HO in ACVR1(R206H)-expressing mice will be analyzed by whole-body imaging and immunohistochemistry to determine the consequence of cell-specific and temporally controlled expression of the mutant receptor. Effects of injury on the occurrence and severity of ACVR1(R206H)-mediated HO also will be evaluated. These studies will define the cellular progenitors and developmental window in which dysregulated intracellular BMP signaling causes HO. Understanding the cellular basis of HO will identify specific cellular targets and suggest therapeutic strategies for HO. Additionally, the mouse models developed herein will be valuable tools for translational studies aimed at developing and testing small molecule therapies and other modalities for the treatment or prevention of HO. PUBLIC HEALTH RELEVANCE: Heterotopic ossification is a painful and debilitating condition characterized by the inappropriate formation of bone in skeletal muscles and associated soft tissues. The proposed studies will identify and characterize the progenitor cell populations that are responsible for heterotopic ossification, thereby defining cellular targets and suggesting therapeutic strategies for the prevention and treatment of this common condition.